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Nitrous oxide abatement coupled with biopolymer production as a model GHG biorefinery for cost-effective climate change mitigation Osvaldo D. Frutos, Irene Cortes, Sara Cantera, Esther Arnaiz, Raquel Lebrero, and Raul Munoz Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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Environmental Science & Technology

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Nitrous oxide abatement coupled with

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biopolymer production as a model GHG

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biorefinery for cost-effective climate change

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mitigation

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Osvaldo D. Frutos†, ‡, Irene Cortes†, Sara Cantera†, Esther Arnaiz†, Raquel lebrero†, Raúl

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Muñoz†*

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† Department of Chemical Engineering and Environmental Technology, University of

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Valladolid, Dr. Mergelina, s/n, 47011, Valladolid, Spain. Tel. +34 983186424

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‡ Facultad de Ciencias Agrarias, Universidad Nacional de Asunción, Campus Ciudad de

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San Lorenzo, Paraguay. Tel. +595 21585606

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*-Author for correspondence: [email protected]

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ABSTRACT

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N2O represents ~6% of the global greenhouse gas emission inventory and the most

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important O3-depleting substance emitted in this 21st century. Despite its environmental

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relevance, little attention has been given to cost-effective and environmentally friendly N2O

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abatement methods. Here we examined, the potential of a bubble column (BCR) and an

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internal loop airlift (ALR) bioreactors of 2.3 L for the abatement of N2O from a nitric acid 1 ACS Paragon Plus Environment


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plant emission. The process was based on the biological reduction of N2O by Paracoccus

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denitrificans using methanol as a carbon/electron source. Two nitrogen limiting strategies

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were also tested for the co-production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

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(PHBV) coupled with N2O reduction. High N2O removal efficiencies (REs) (≈87%)

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together with a low PHBV cell accumulation were observed in both bioreactors in excess of

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nitrogen. However, PHBV contents of 38-64% were recorded under N limiting conditions

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along with N2O-REs of ≈57% and ≈84% in the ALR and BCR, respectively. Fluorescence

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in-situ hybridization analyses showed that P. denitrificans was dominant (>50%) after 6

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months of experimentation. The successful abatement of N2O concomitant with PHBV

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accumulation confirmed the potential of integrating biorefinery concepts into biological gas

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treatment for a cost-effective GHG mitigation.

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TOC/Abstract Art

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INTRODUCTION

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The increasing global temperature rise and climate change reported by scientists has

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attracted the attention of the community and the policy makers during the past decade.

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Nowadays, there is now good evidence that these environmental problems are caused by

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the rapid accumulation of greenhouse gases (GHGs), whose concentrations are 45 % higher

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than those prevailing in the preindustrial era.1 Nitrous oxide (N2O), the third most

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important GHG with a global warming potential 300 times higher than that of CO2 (on a

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mol per mol basis) due to its high atmospheric persistence (150 years), accounts for 6.2 %

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of the total GHG emissions globally. N2O is also one of the main sources of stratospheric

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NOx and is considered the most important ozone depleting substance emitted in this 21st

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century.2 Agriculture is the principal source of anthropogenic N2O emissions, followed by

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chemical industry and waste management processes. The production of nitric and adipic

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acid are the major N2O source in industry, whose global emissions reach up to 400 Kton of

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N2O per year.3 A typical waste gas from nitric acid production plants is characterized by

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100-3500 ppmv of NOx, 300-3500 ppmv of N2O, 1-4 % of O2 and 0.3-2 % of H2O (in a N2

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matrix).4

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Several physical-chemical technologies such as non-selective catalytic reduction (NSCR)

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or catalytic decomposition have been applied as end-of-the-pipe strategies for the treatment

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of N2O emissions from industrial sources.5 However, these technologies entail the

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consumption of a reducing agent such as hydrocarbons or ammonia and require the

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preheating of the tail gas for N2O destruction, resulting in a considerable energy

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consumption since nitric acid waste gas is typically emitted at ambient temperature.6

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Moreover, the environmental sustainability of NSCR technologies can also lead to 4 ACS Paragon Plus Environment

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production of CH4, another atmospheric GHG, derived from an incomplete fuel combustion

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during the treatment of N2O.7

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Biological technologies have been shown to exhibit a high robustness, cost efficiency and

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environmental friendliness for the treatment of industrial off-gases containing malodorous

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and volatile organic compounds.8 In spite of their inherent advantages, no biological

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process has ever been evaluated for the abatement of N2O emissions from nitric and adipic

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acid plants.9,10 This GHG is an obligate intermediate in the reduction of NO3- and NO2- to

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N2, which to the best of our knowledge is the only known biochemical mechanism for N2O

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removal. Thus, since nitric and adipic acid emissions are mainly composed of N2O, N2 and

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trace levels of O2, denitrification appears as an attractive alternative for the abatement of

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N2O when a cheap source of organic carbon and electron donor is available for the growth

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of heterotrophic bacteria.9,10 In this context, the economic viability of this process can be

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significantly improved by coupling the abatement of N2O via denitrification with the

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production of added value bioproducts such as polyhydroxyalkanoates (PHA) biopolymers.

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These bio-based chemicals, especially poly(3-hydroxybutyrate) (PHB) and poly(3-

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hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), share with conventional fossil-derived

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thermoplastics similar physical/chemical characteristics such as melting point, molecular

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weight and tensile strength.11 PHAs also possess a rapid biodegradability in nature, which

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render them a perfect substitute of conventional fossil polymers. There are several

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denitrifying bacteria such as Paracoccus denitrificans, Pseudomonas aeruginosa and

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Ralstonia eutropha, capable of producing intracellular PHA as a carbon storage material in

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excess of organic carbon under nutrient limitation.12,13 Therefore, an innovative GHG

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biorefinery could be engineered for the simultaneous abatement of N2O and co-production 5 ACS Paragon Plus Environment


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of PHAs in nitric and adipic acid plants in order to enhance the economic and

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environmental sustainability of N2O abatement.

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In this context, the performance of two low-cost and eco-friendly suspended-growth

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bioreactors, namely bubble column (BCR) and airlift (ALR), was evaluated for the

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treatment of a synthetic N2O emission from nitric acid plant. The strain Paracoccus

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denitrificans (DSM 413) was used as a model denitrifying bacterium in the co-production

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of the co-polyester PHBV using methanol as a carbon-energy source under nitrogen

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sufficiency and two different nitrogen limiting strategies.

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MATERIALS AND METHODS

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Chemicals and mineral salt medium

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The mineral salt medium (MSM) used in the experimentation was composed of (g L-1):

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Na2HPO4·12H2O 6.16, KH2PO4 1.52, MgSO4·7H2O 0.2, CaCl2 0.02, NH4Cl 1.5 and 10 mL

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L-1 of a trace element solution containing (g L-1): EDTA 0.5, FeSO4·7H2O 0.2,

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ZnSO4·7H2O 0.01, MnCl2·4H2O 0.003, H3BO3 0.03, CoCl2·6H2O 0.02, CuCl2·2H2O 0.001,

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NiCl2·6H2O 0.002, NaMoO4·2H2O 0.003. The final pH of the MSM was 7. All reagents,

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including methanol, were purchased from PANREAC with a purity of >99 %. Benzoic acid

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(>99 %) and PHBV standards were obtained from Sigma-Aldrich® (Sigma-Aldrich, St.

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Louis, MO, USA). A 40 L calibrated gas cylinder of 50,000 ppmv of N2O in N2 and 50 L

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industrial N2 cylinder were purchased from Abelló Linde S.A. (Barcelona, Spain).

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Microorganism cultivation


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The methylotrophic strain Paracoccus denitrificans (DSM 413) was purchased from

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DSMZ (Braunschweig, Germany). The bacterium was cultivated in sterilized 1 L E-flasks

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with 0.5 L of MSM with methanol (1 % v/v) as the sole carbon and energy source under

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aerobic conditions for 3 weeks.

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Experimental set up

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A BCR of 42 cm of height (H) and 9 cm of inner diameter (ID), and an ALR of the same

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dimensions with a concentric draft tube (ID = 5.5 cm, H = 29.5 cm) located at 4 cm from

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the bottom of the reactor, were inoculated with 0.5 L of P. denitrificans inoculum and filled

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with MSM to a working volume of 2.3 L, resulting in an initial total suspended solid (TSS)

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concentration of 56 mg L-1 in both bioreactors. The synthetic nitric acid plant N2O emission

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was obtained by mixing the calibrated mixture of N2O (50,000 ppmv), air from a

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compressor and pure N2 using mass flow controllers (Aalborg, Denmark). The gas mixture

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resulted in BCR and ALR inlet N2O gas concentrations of 3520 ± 290 and 3560 ± 300

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ppmv, respectively. The O2 inlet gas concentration remained at 1.1 ± 0.1 % in each

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bioreactor. Both the BCR and ALR were supplied with an inlet gas flow rate of 137 ± 8 and

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140 ± 10 mL min-1, respectively, which corresponded to a gas empty bed residence time

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(EBRT) of ≈17 min. Pure methanol (CH3OH) was injected in the gas line by means of a

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syringe pump in a sample port filled with fiberglass wool to facilitate solvent evaporation.

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Methanol was selected as a carbon and energy source based on its empirically proven

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ability to support biopolymer accumulation in P. denitrificans, its low cost and extensive

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use in wastewater treatment plants for denitrification purposes.14 The systems were

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operated in a controlled temperature room at 25 ºC. A detailed diagram of the experimental

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setup can be found in Figure S1 (supporting information). 7 ACS Paragon Plus Environment


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Operational conditions

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Three operational strategies, corresponding to Stages I, II, and III, were evaluated in both

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bioreactors under different MSM nitrogen concentrations in order to assess the feasibility of

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a simultaneous N2O removal and PHBV cell accumulation. During the first 43 days of

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operation (Stage I) the bioreactors were maintained under nitrogen sufficiency to evaluate

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both the N2O removal performance and the accumulation of PHBV under optimal

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cultivation conditions by supplying MSM with 396 mg N L-1 and 124 g C m-3 d-1 of

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CH3OH. During Stage I, 300 mL of the cultivation broth was replaced by fresh MSM three

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times per week, which resulted in a dilution rate of ̴ 0.056 d-1 and an N inlet load of 22.1 g

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N m-3 d-1. Stage II (days 44 to 127) was devised to promote the accumulation of

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intracellular PHBV at a CH3OH inlet load of 93 g C m-3 d-1, which guaranteed carbon

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availability. The N concentration in the MSM was reduced to 34 mg N L-1 during Stage II,

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with 300 mL of fresh MSM being replaced every two days. This resulted in a N inlet load

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of 2.2 g N m-3 d-1, a dilution rate of 0.065 d-1 and nitrogen fast:famine cycles of 1d:1d . In

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Stage III (days 128 to 179), the nitrogen concentration in the MSM was increased to 68 mg

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N L-1 while decreasing the frequency of MSM replacement (300 mL) from two to four days

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at a CH3OH inlet load of 108 g C m-3 d-1. The dilution rate and N inlet load during Stage III

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was 0.033 d-1 and 2.2 g N m-3 d-1. The strategy conducted in this last stage aimed at

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increasing the biomass concentration in the bioreactors and consequently the N2O removal

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performance by simultaneously reducing and increasing the dilution rate and N-MSM

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concentration, respectively. A mass transfer test was carried out according to Cantera and

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coworkers15 at the end of Stages II and III by increasing the N2O inlet concentration from


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≈3527 to ≈9058 in order to elucidate the limiting factor during N2O reduction to N2 under

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the experimental conditions evaluated.

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Sampling and analytical procedures

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The gas phase monitoring procedure entailed the periodical measurement of N2O, CO2 and

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O2 gas concentrations at both inlet and outlet bioreactors sampling ports. The monitoring of

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the liquid phase involved the withdrawal of 300 mL of cultivation broth from each

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bioreactor in order to determine the dissolved total organic carbon (TOC), total nitrogen

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(TN), CH3OH, TSS and PHBV concentrations. The dissolved oxygen concentration was

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measured in-situ. In addition, 20 mL of the cultivation broth was centrifuged, wash with

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distilled water and dried at 105 ºC for 24 h for the measurement of C, N, H and S cell

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content at the end of each experimental conditions.

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The N2O and CO2/O2 gas concentrations were measured by GC-ECD and GC-TCD

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according to Frutos et al.9 and Lopez et al.,16 respectively. TOC and TN concentrations

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were measured using a TOC-VCSH analyzer (Shimadzu, Tokyo, Japan) coupled with a

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total nitrogen chemiluminescence detection module (TNM-1, Shimadzu, Japan). Dissolved

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CH3OH concentration was determined in a GC-FID (Bruker 3900, Palo Alto, USA)

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equipped with a SupelcoWax (15 m × 0.25 mm × 0.25 µm) capillary column. GC-FID

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injector and detector temperatures were maintained at 200 and 250 ºC, respectively.

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Nitrogen was used as the carrier gas at 1 mL min-1 while H2 and air flows were fixed at 30

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and 300 mL min-1, respectively. N2 was also used as the make-up gas at 25 mL min-1. The

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determination of TSS concentration was performed according to standard methods17. The

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dissolved oxygen concentration was measured with a handheld OXI 330i oximeter (WTW,



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Germany) while pH was periodically monitored using a pH/mV/°C meter (pH 510 Eutech

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Instruments, Nijkerk, the Netherlands). To quantify the PHBV concentration, 2 mL of the

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cultivation broth were centrifuged at 9000 rpm for 15 min and the biomass pellet obtained

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was processed according to Zuñiga and coworkers,18 using chloroform as extraction

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solvent. The PHBV extracted was measured by GC-MS (Agilent Technologies: GC System

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7820A MSD 5977E, Santa Clara, USA) equipped with a DB-wax column (30 m × 250 µm

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× 0.25 µm) with detector and injector temperatures of 250 ºC and a split ratio of 1:10. The

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oven temperature was initially maintained at 40 ºC for 5 min, increased at 10 ºC min-1 up to

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200 ºC and maintained at this temperature for 5 min. The PHBV cell content was

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normalized as %PHBV= (g PHBV/g TSS) × 100. The analysis of C, N, H and S biomass

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content was conducted using a LECO CHNS-932 elemental analyzer.

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Electron microscopy analysis

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Cultivation broth samples of 1 mL were drawn in order to assess the presence of PHBV

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inside the cells by microscopy analysis at the end of Stage III. The samples were

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centrifuged at 4000 rpm and 4 ºC for 5 min, to subsequently process the biomass pellets

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according to Bozzola.19 The samples were then cut in thin slices by a microtome and

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contrasted according to Wendlandt and coworkers.20 A TEM JEOL JEM-1011 electron

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microscope (Teknolab, Indonesia) equipped with an ES1000W Erlangshen CCD camera

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(Gatan, Germany) was used for the analysis.

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Fluorescence in situ hybridization (FISH) analysis

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FISH analyses were conducted to evaluate the abundance of P. denitrificans in the culture

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broth during each operational stage since the systems were run under non-sterile conditions 10 ACS Paragon Plus Environment

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(similarly to the scenario envisaged for its large scale implementation). Aliquots of 250 uL

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of the cultivation broth from both bioreactors at the end of each operational stage were

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fixed in 4 % (w/v) paraformaldehyde for 3 h, washed three times with phosphate-buffered

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saline (PBS) and then preserved in alcohol 96 % (v/v). Aliquots of 10 µL of samples were

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placed on glass microscope slides and dehydrated with ethanol at 50 %, 80 % and 96 %

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(v/v). The probes used were EUB338 I-II-FITC (for general bacteria)21,22 and PAR651-Fam

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(specific for the genus Paracoccus).23 Hybridization was carried out at 46 ºC using

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formamide at 40 % .24 For quantitative FISH analysis, 16 images were randomly acquired

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from each well on the slides using a Leica DM4000B microscope (Leica Microsystems,

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Wetzlar, Germany). The relative bio-volumes of the specific genus Paracoccus from the

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total bacteria (EUB338 I-II) were calculated using the commercial software DAIME and

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split into individual color channels before image segmentation.25

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RESULTS AND DISCUSSION

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Influence of nitrogen supplementation on N2O abatement

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The two bioreactors exhibited a low and stable dissolved oxygen (DO) concentration during

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the entire experimentation (0.07 ± 0.1 mg L-1 in both bioreactors). Similarly, a stable pH of

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6.8 ± 0.2 was recorded in both systems along the three operational stages. The N2O REs

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reached a steady state 10 days after the startup of the bioreactors. Hence, steady state N2O

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REs of 87 ± 3 % were reached during Stage I in the BCR with inlet and outlet N2O

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concentrations of 3380 ± 340 and 440 ± 74 ppmv, respectively (Figure 1A). Similarly, the

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ALR supported steady state REs of 88 ± 2 % with inlet and outlet N2O concentrations of

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3610 ± 340 and 420 ± 69 ppmv, respectively (Figure 1B).



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Figure 1. Time course of the inlet (□) and outlet (○) N2O gas concentrations and removal

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efficiency (solid line) in the BCR (A) and ALR (B). Vertical lines indicate the different

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operation stages.

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The CO2 produced from the oxidation of CH3OH during Stage I was correlated with the

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removal of N2O, resulting in comparable CO2 production rates of 85 ± 8 g C m-3 d-1 and 91

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± 8 g C m-3 d-1 in the BCR and ALR, respectively (Figure 2A). In this context, the ratio of

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C-CO2 produced per C-CH3OH consumed in this steady state accounted for 0.73 and 0.78

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C-CO2 C-CH3OH-1 in the BCR and ALR, respectively. Biomass concentration, measured as

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TSS, reached stable values of 853 ± 76 and 856 ± 90 mg L-1 in BCR and ALR, respectively,


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after 20 days of operation (Figure 2B). These biomass concentrations entailed specific N2O

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removal capacities of 0.52 ± 0.07 gN2O gTSS-1 d-1 in the BCR and 0.66 ± 0.08 gN2O gTSS-

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1

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in both bioreactors, with average values of 0.7 gCO2 gN2O-1 in the BCR and 0.6 gCO2

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gN2O-1 in the ALR.

d-1 in the ALR. Likewise, the ratio of CO2 produced per mol of N2O reduced were similar

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Figure 2. Time course of CO2 production rates (A) and TSS concentrations (B) in the BCR

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(∆) and ALR (○). Vertical lines indicate the different operation stages.

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The decrease in N supply rate from day 44 (Stage II) in order to achieve 1d:1d nitrogen

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fast-famine cycles resulted in a progressive reduction in the N concentration down to a 13 ACS Paragon Plus Environment


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complete depletion by day 66 in both bioreactors (Figure 3). Nitrogen depletion entailed a

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gradual deterioration in N2O REs down to steady state values of 62 ± 7 % in BCR and 58 ±

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6 % in ALR (Figure 1A). This significant decrease in N2O REs was correlated to a

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concomitant reduction in biomass concentration as a result of the limited N availability. In

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this context, the TSS concentration decreased gradually to steady values of 422 ± 76 in the

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BCR and of 285 ± 99 mg L-1 in the ALR from day 94 (Figure 2B). Surprisingly, the

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microbial population in the ALR was more impacted by N deprivation than that present in

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the BCR. Furthermore, the presence of the internal draft tube in the ALR likely entailed

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higher gas bubble rising velocities compared to those in the BCR, and thus a lower N2O

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mass transfer to the liquid phase that limited biomass growth. In addition, biofilm

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formation in the internal draft tube of the ALR could have led to an underestimation of the

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TSS concentrations. The reduction in biomass concentration and N2O RE resulted in a

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concomitant decrease in the CO2 production rate in the ALR (63 ± 3 g C m-3 d-1) compared

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to CO2 production rates of 78 ± 7 g C m-3 d-1 in the BCR (Figure 2A). This reduction

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resulted in an increase in the CO2 produced per N2O consumed ratio up to 0.83 and 0.75

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gCO2 gN2O-1 in the BCR and ALR, respectively. Similarly, an increase in the CO2 produced

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per CH3OH consumed ratio was observed in both bioreactors (0.99 and 0.96 C-CO2 C-

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CH3OH-1, respectively), which indicated that cell maintenance increased as a result of N

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deprivation. A mass transfer test was conducted at this point to assess the limiting factor in

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N2O removal during Stage II. An increase in the N2O inlet load by a factor of 2.4 ± 0.2 did

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not result in a concomitant increase in CO2 production rate and N2O elimination capacity

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(Figure S2). Hence, this test confirmed that both bioreactors were limited by microbial

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activity due to the low biomass concentration supported by the limited N supply imposed.

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Furthermore, the highest specific N2O removal were recorded during Stage II, with average 14 ACS Paragon Plus Environment

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values of 0.76 ± 0.19 gN2O gTSS-1 d-1 in the BCR and 1.31 ± 0.34 gN2O gTSS-1 d-1 in the

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ALR.

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The increase in N concentration by a factor of 2 along with the reduction in the dilution rate

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from 0.065 d-1 to 0.033 d-1 by day 127 (Stage III) supported an increase in biomass

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concentration to 1017 ± 71 mg TSS L-1 and 646 ± 64 mg TSS L-1 in the BCR and ALR,

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respectively. This entailed a concomitant increase of N2O removal in the BCR up to steady

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REs of 84 ± 3 % but similar N2O-REs of 57 ± 7 % were recorded in the ALR during Stage

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III (Figure 1, Figure 2). This increase in biomass concentration did support an increase in

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the CO2 production rates up to 85 ± 5 and 78 ± 6 g C m-3 d-1 in the BCR and ALR,

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respectively. The CO2 produced per N2O consumed ratio showed a slight increase in the

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ALR (0.72 gCO2 gN2O-1) and a decrease to 0.66 gCO2 gN2O-1 in the BCR. On the other

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hand, the CO2 produced per CH3OH consumed ratio decreased to 0.81 and 0.85 C-CO2 C-

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CH3OH-1 in the BCR and ALR, respectively, due the enhanced biomass growth. In this

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context, the mass transfer test carried out at the end of Stage III revealed that both systems

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were limited by N2O mass transfer. Thus, the increase in N2O inlet load by a factor of 2.8 ±

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0.1 promoted a rapid increase in the N2O elimination capacity by a factor of 2.4 ± 0.1 in

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both reactors (Figures S3). Mass transfer limitations have been previously identified as the

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limiting step in a bioscrubber treating N2O laden air emissions from wastewater treatment

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plants, where a gas EBRT of 40 min was needed in the adsorption column in order to

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obtain a satisfactory N2O RE of 92 %.10 Indeed, previous studies have consistently shown

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gas-liquid mass transfer limitations in bioreactors devoted to the treatment of poorly water

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soluble gas pollutants.15,26 This mass transfer limitation could be also observed in the

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decrease of the specific N2O removal capacity of both bioreactors, which resulted in 15 ACS Paragon Plus Environment


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average values of 0.47 ± 0.03 and 0.54 ± 0.1 gN2O gTSS-1 d-1 in the BCR and ALR,

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respectively. This limited N2O mass transfer from the gas emission entails process

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operation at high EBRTs, thus resulting in large bioreactor volumes (= high investment

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costs). In this sense, the use of ceramic or polymer membrane gas diffusers with a pore

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size < 1 µm and internal gas recycling strategies can significantly enhance the volumetric

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gas-liquid mass transfer coefficient (at the expenses of slightly higher operating costs).27,28

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Another potential alternative to overcome this mass transfer limitation is the addition to the

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bioreactor of an immiscible, non-volatile, non-toxic and non-biodegradable non-aqueous

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phase (NAP) with a high affinity for N2O.27,29 However, the selection of the optimum

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NAPs to enhance the mass transfer of N2O in the so called two-phase partitioning

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bioreactors has not been conducted yet.

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This study evaluated the potential biological removal of N2O along with the simultaneous

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accumulation of PHBV as a cost-effective alternative for industrial GHG emission

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mitigation. The bioreactors herein used have been consistently proven as low cost

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alternative technologies for the treatment of wastewaters and off-gases.30–33 These

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bioreactor configurations are pneumatically agitated, resulting in low energy consumptions.

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Moreover, their simple construction (with no moving parts) and high gas-liquid mass

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transfer rates constitute also key advantages over their biological counterpart.34–36 In

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addition, the selection of BCR and ALR configurations in this study was based on the need

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of a suspended culture to facilitate biomass harvesting and ensure a homogeneous

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methanol/nitrogen distribution. In our particular study, N2O-REs of 80-90 % were

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consistently achieved concomitantly with the co-production of added value biopolymers

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(see section below), which were comparable with the N2O abatement efficiencies of 16 ACS Paragon Plus Environment

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conventional physical/chemical technologies such as NSCR7. Decrease in the performance

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of N2O removal was observed during Stage II due the low biomass concentrations, but was

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easily overcame with the strategy implemented in Stage III (same nutrient load at a higher

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inlet N concentration) maintaining a high biopolymer accumulation (Figure 1A and 3A).

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However, the gas EBRT (≈17 min) required to obtain high REs in the two bioreactor

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configurations evaluated would entail high bioreactor volumes.

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PHBV accumulation during N2O abatement

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A low PHBV cell content was recorded during Stage I (1.9 ± 1.3 % in the BCR and 2.6 ±

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1.3 % in the ALR) under TN concentrations in the cultivation broth of 238 ± 38 and 238 ±

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40 mg N L-1 in the BCR and ALR, respectively. The dissolved CH3OH concentrations in

308

the BCR and ALR also remained constant during Stage I at 395 ± 20 and 367 ± 39 mg C L-

309

1

, respectively.



Page 18 of 28

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311

Figure 3. Time course of the PHBV cell content (∆) and TN concentrations (○) in the BCR

312

(A) and the ALR (B). Vertical lines indicate the different operation stages.

313

N was completely depleted by day 66 in Stage II, which promoted the gradual increase in

314

the PHBV cell content in both bioreactors (Figure 3). The nitrogen supply strategy

315

evaluated during Stage II resulted in 24 hour of nitrogen sufficiency after MSM renewal

316

followed by 24 h under nitrogen limitation, where PHBV synthesis and accumulation was

317

likely to occur. N-limitation induced a steady state PHBV cell content of 38 ± 7 % in the

318

BCR under TN and dissolved CH3OH concentrations of 2.6 ± 0.5 mg N L-1 (Figure 3A) and

319

177 ± 28 mg C L-1, respectively. The PHBV cell content recorded in the ALR was

320

significantly higher than in the BCR, with average values of 64 ± 11 % (Figure 3B) under 18 ACS Paragon Plus Environment

Page 19 of 28


321

steady TN concentrations of 2.8 ± 0.6 mg N L-1 and dissolved CH3OH concentrations of

322

368 ± 39 mg C L-1. The transmission electron micrographs depicted in Figure 4 confirmed

323

the accumulation of PHBV as granules inside bacteria with a cell diameter ranging from 0.5

324

to 1 µm, which matched the cell size of P. denitrificans.37

325 326

Figure 4. Transmission electron micrographs of cells containing PHBV in the BCR (a, c)

327

and the ALR (b). Samples were drawn at the end of Stage III.

328

Process operation at a reduced dilution rate of 0.033 d-1 under similar N loads as Stage II

329

mediated microbial cultivation with N sufficiency for 24 h followed by 3 days of N

330

deprivation in Stage III. These operational conditions promoted an enhanced PHBV cell

331

content in the BCR of 47 ± 5 %, under steady TN and dissolved CH3OH concentrations of

332

2.3 ± 0.3 mg N L-1 and 134 ± 23 mg C L-1, respectively. However, the PHBV cell content

333

recorded in the ALR decreased to 40 ± 8 % in spite of the comparable TN concentration

334

(1.9 ± 0.4 mg N L-1) and dissolved CH3OH concentrations (373 ± 72 mg C L-1). Both

335

bioreactors supported low PHBV productivities in excess of nitrogen (~0.8 g PHBV m-3 d19 ACS Paragon Plus Environment


Page 20 of 28

336

1

). These productivities increased in Stage II up to 10 g PHBV m-3 d-1 in both bioreactors.

337

The highest PHBV productivities were recorded in the last stage of BCR operation (15 g

338

PHBV m-3 d-1), although PHBV productivity in the ALR decreased to 7 g PHBV m-3 d-1 in

339

stage III. PHBV yield of 0.06 ± 0.02 gPHBV g-1CH3OH was observed in the ALR in Stage II,

340

decreasing to average values of 0.03 ± 0.01 gPHBV g-1CH3OH during Stage III. The BCR

341

supported comparable production yields of 0.05 ± 0.01 gPHBV g-1CH3OH and 0.06 ± 0.02

342

gPHBV g-1CH3OH in Stages II and III, respectively. The yields obtained were in agreement

343

with previously reported PHBV yields ranging from 0.02 to 0.19 gPHBV g-1CH3OH using

344

methanol as the carbon source.38–40

345

The GC-MS analysis of the copolymer PHBV showed a small share of 3-hydroxyvalerate

346

(PHV) regardless of the operational conditions evaluated. PHV/PHBV molar ratios of 2.5 ±

347

0.9 % and 2.9 ± 1.6 % were recorded at Stage I in the BCR and the ALR, respectively.

348

When the bioreactors were subjected to nutrient limitation during Stages II and III, this

349

ratio decreased to 0.46 ± 0.2 and 0.29 ± 0.1 in the BCR, and to 0.35 ± 0.1 and 0.32 ± 0.2 %

350

in the ALR, respectively. Several authors have recorded similar results using methanol as

351

the sole carbon and energy source under different nutrient limitation strategies. In this

352

context, Ueda et al.41 did not detect PHV in the PHBV copolymer accumulated in P.

353

denitrificans when CH3OH was used as the sole substrate (0.3% v/v). However, the PHV

354

molar fraction increased up to 87 % when n-amyl alcohol (0.25 % v/v) was supplied

355

together with CH3OH (0.3 % v/v) . Similarly, Yamane et al.12 explored the role of the type

356

of alcohols (methanol, ethanol, n-propanol, n-butanol and n-pentanol) at a concentration of

357

0.1 % (v/v) on the PHBV cell content in P. denitrificans under N limiting conditions. The

358

results revealed no PHV accumulation when CH3OH was the sole carbon source, which 20 ACS Paragon Plus Environment

Page 21 of 28


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suggested that CH3OH is not the most suitable carbon source when a high share of PHV is

360

desired. In spite of the non-negligible cost associated to the purchase of the methanol (≈180

361

€ ton-1)14 required for biological N2O reduction, this operating cost will be compensated by

362

the fact that a large fraction of this methanol is converted into PHBV (with a market price

363

of 4000-2000 € ton-1)

364

The analysis of the elemental cell composition (C, H, S, and N) carried out at the end of

365

Stage I showed C and N cell contents of ≈44 and ≈11 %, respectively (Table S1), which

366

represented a C/N ratio of ≈4. This value was in agreement with the typical elemental

367

composition for bacterial cells.42 However, a significant reduction in N cell content was

368

observed in the biomass from both bioreactors as a result of cell adaptation when N limiting

369

strategies were implemented in Stages II and III. Thus, the C/N ratio recorded in the ALR

370

and the BCR under nitrogen limitation increased to values ranging from 6.1 to 8.2. The

371

likely decrease in protein cell content due to the limited N uptake also entailed a decrease in

372

the S content of the microbial communities present in both bioreactors. A variation in the C

373

cell content was not observed in spite of the accumulation of the biopolymer likely due to

374

the similar elemental composition (C, H and O) of PHBV and biomass.

375

FISH analysis of the microbial population structure

376

The FISH analysis revealed the variation of the abundance of the P. denitrificans along the

377

entire operational period (Table S2). Both bioreactors showed a P. denitrificans abundance

378

higher than 90 % by the end of Stage I (Figure 5, Table S2). At the end of Stage II (day

379

120), the abundance of the inoculated strain in the BCR and ALR slightly decreased to 88

380

% and 86 % (Table S2). These results confirmed that P. denitrificans was capable of



Page 22 of 28

381

growing and dominating the microbial culture under anoxic conditions using CH3OH as the

382

sole carbon/energy source and N2O as electron acceptor. By the end of the experimentation

383

(day 180), P. denitrificans remained dominant in both bioreactors (abundances > 50 %)

384

(Figures 5c and 5f). In this context, the presence of others microbial strains capable of

385

accumulating biopolymers may explain the maintenance of the PHBV cell content observed

386

in Stage III despite the decrease in P. denitrificans abundance. Nonetheless, it is important

387

to point out that FISH analysis could have slightly underestimated the abundances of P.

388

denitrificans due to the foreground effect, which prevented the observation of the bacterial

389

cells present in the backside of the flocs.

390

391

Figure 5. FISH micrographs of the microbial culture present at the end of the three

392

operational stages evaluated in the ALR (a-c) and BCR (d-f). PAR651-fam (green) appears

393

yellow due to a double hybridization with the EUB338 I-II- FITC probes (red).

394

In summary, this work demonstrated the feasibility of the combined biological abatement

395

of N2O from industrial emissions and co-production of PHBV. High N2O-REs were

396

recorded in spite of process operation under nitrogen limiting conditions. The nitrogen 22 ACS Paragon Plus Environment

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limiting strategies assessed in this study resulted in a high accumulation of PHBV by P.

398

denitrificans using methanol and N2O as the carbon/energy source and the electron

399

acceptor, respectively. This study reports the first bioprocess for the active abatement of

400

N2O using a waste-to-value biorefinery approach.

401

ASSOCIATED CONTENT

402

Supporting Information

403

A file with additional information is available free of charge via internet at

404

http://pubs.acs.org. This file includes a schematic diagram of the operational set-up

405

depicted in Figure S1 as well as additional data obtained from the mass transfer tests

406

conducted at the end of Stages II (Figure S2) and III (Figure S3). Furthermore, the results

407

from the analysis of the elemental composition (Table S1) and FISH analysis (Table S2) of

408

the biomass are included in the file.

409

AUTHOR INFORMATION

410

Corresponding Author

411

*Phone: 0034983186424; fax: 0034983423013; e-mail: [email protected].

412

Present Addresses

413

┴R.M.: University of Valladolid, Dr. Mergelina s/n, Valladolid 47011, Spain.

414

Notes

415

The authors declare no competing financial interest.

416

ACKNOWLEDGMENT 23 ACS Paragon Plus Environment


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This research was supported by the Spanish Ministry of Economy and Competitiveness

418

(CTM2015-70442-R and Red NOVEDAR CTQ2014-51693-REDC projects), the regional

419

government of Castilla y León (UIC 71) and the European Commission through the

420

Erasmus Mundus Program BABEL and FEDER Funding Program.

421

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